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Treatments for contaminant reduction in lactoferrin preparations and lactoferrin containing compositions

a technology of lactoferrin and composition, which is applied in the direction of transferrins, peptides/protein ingredients, peptides, etc., can solve the problems of limiting the usefulness of lf protein, further compromising lf applications, and adversely affecting lf bioactivity, so as to achieve significant reduction and/or elimination of viable microbial load and endotoxin activity, and enhance health benefits

Active Publication Date: 2005-09-08
NAIDU LP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a method for reducing contaminants in commercial lactoferrin preparations, resulting in a purified lactoferrin with reduced viable microbial load and endotoxin activity. The method involves treating the commercial lactoferrin with a surfactant and an antioxidant, followed by purification using polyphenols and an anionic compound. The ultra-cleansed lactoferrin preparation can be used as a physiologic delivery system to enhance health benefits in humans and can also contain other ingredients such as surfactants, antioxidants, polyphenols, and anionic compounds. The therapeutic lactoferrin composition can be used for various applications such as prebiotics, nutraceuticals, and probiotics."

Problems solved by technology

There are four major issues critical for the commercialization of LF as a prebiotic in vivo delivery system including bioactivity, microbiological quality, endotoxin content and dosage.
Thus, the stability of LF protein could limit its usefulness.
Furthermore, protein isolation and processing conditions including storage, freezing / thawing, and spray-drying could also adversely affect LF bioactivity.
Degradation of native LF into peptic fragments as well as the co-elution of impurities from raw material could further compromise LF applications.
Therefore, LF could denature or inactivate to partially or totally lose its functional properties during large-scale manufacturing.
However, immobilized lactoferrin is neither designed nor is reported to have prebiotic activity.
The microbiological quality of LF starting raw materials could significantly compromise the human health applications of commercial LF.
Similarly, microbiological quality issues also exist for genetically modified organism (GMO)-derived and recombinant LFs from various expression sources such as rice, tobacco, yeast, cell cultures or transgenic animals.
The endotoxin content of LF starting raw material could also adversely affect its human health applications.
Thus, the microbiological standards of chromatographic resins, sanitation practices of processing equipment and even more significantly the water quality used in LF isolation, could cumulatively contribute to the endotoxin levels of the isolated LF material and thereby could limit the in vivo applications of commercial LF.
In such abnormal conditions, LF aggregates into dimeric and tetrameric complexes that could subsequently lead to LF dysfunctionality.

Method used

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  • Treatments for contaminant reduction in lactoferrin preparations and lactoferrin containing compositions
  • Treatments for contaminant reduction in lactoferrin preparations and lactoferrin containing compositions

Examples

Experimental program
Comparison scheme
Effect test

example 1

TCR Process

[0140] A 1% (v / v) polysorbate-80 solution, a 0.5% solution of vitamin C (as calcium-L-ascorbate) with 10 mM sodium bicarbonate and 95% (w / v) turmeric root extract (0.1% curcumin) were used in the TCR process as surfactant, antioxidant and polyphenolic tiers, respectively.

[0141] Experiment 1:

[0142] Ten grams of commercial whey-derived LF powder with a bioburden of 7200 CFU / g was dissolved in 100 mL of polysorbate-80 solution with gentle stirring at room temperature for 2 h. Quadruplicate samples of 10 mL each were aspirated from the homogenate and tested for bioburden according to the BAM protocol. The polysorbate-80 surfactant in the presence of LF caused about a 35% reduction in aerobic plate count (APC) and 40% reduction in yeast / mold counts. However, in this solution the coliform counts and the endotoxin activity in the LF remained unaffected.

[0143] Experiment 2:

[0144] Ten minutes after adding 10 g LF to polysorbate-80 (100 mL) the solution was mixed with calcium-...

example 2

Evaluation of TCR Process on Different Types of LF

[0148] The efficacy and possible broad-scale application of the TCR process to reduce the bioburden from different commercial types of LF (bovine LF derived from milk and whey; recombinant human LF expressed in a plant system) was evaluated. The TCR process consisted of the three tier system and the experiments were performed as described in Example 1. The TCR process results were expressed as cumulative endpoint data, consolidating the contaminant reductions in all three tiers.

[0149] In addition to treating the intrinsic contaminant loads, the efficacy of the TCR process was also tested against LF preparations challenged with specific pathogens. Accordingly, the milk- and whey-derived LF were challenged with an APC cocktail (˜5-log CFU / g) consisting of E. coli ATCC43895 (3-log CFU / g), Staph. aureus ATCC25923 (3.5-log CFU / g) and Listeria monocytogenes ATCC191 15 (3.2-log CFU / g) and subjected to the TCR process.

[0150] Table 3 summa...

example 3

Functional Analysis for Prebiotic Activity of LF after TCR

[0153] The formulation composition is known to exert a significant effect on the structure-functional properties of LF. Therefore, the effects of the TCR process on the multifunctional activities of LF was evaluated. Accordingly, a functional analysis was performed with LF after TCR (LF-TCR) with respect to its prebiotic effects on lactic acid bacteria, antioxidant activity in combination with components of the TCR process and safety / toxicity by measuring cellular apoptosis upon exposure. The functional analysis data was compared with LF preparations prior to the TCR process, i.e. untreated controls.

[0154] The growth-multiplication of 18 different probiotic LAB (including three strains of Bifidobacterium spp., 13 strains of Lactobacillus spp., one strain each of Lactococcus spp., and Streptococcus spp.) was measured in the presence of LF-TCR and compared with untreated LF and controls (without any LF exposure). Two differen...

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Abstract

A method of preparing an ultra-cleansed lactoferrin preparation, termed treatment for contaminant reduction (TCR) is provided which includes the steps of treating commercial lactoferrin preparation with at least one each of surfactants, antioxidants and polyphenols to form purified lactoferrin (LF-TCR) and drying the LF-TCR. Additionally a therapeutic lactoferrin composition is provided which contains LF-TCR and optionally surfactants, antioxidants, polyphenols, tissue / membrane diffusion facilitating agents and anionic compounds. The therapeutic lactoferrin composition can additionally contain bioactive agents, dietary supplements, nutraceuticals / functional foods, prophylactic agents, therapeutic agents and probiotic lactic acid bacteria.

Description

RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Patent Application No. 60 / 549,998 filed Mar. 3, 2004.FIELD OF THE INVENTION [0002] This invention relates to a method of ultra-cleansing preparations of commercially available lactoferrin (LF) to render them suitable for prebiotic as well as other functional in vivo applications. Additionally the invention relates to physiological delivery systems for prebiotic lactoferrin compositions with or without supplementation with probiotic lactic acid bacteria and other bioactive compounds. BACKGROUND OF THE INVENTION [0003] The emerging knowledge on diseases and the role of natural foods in lowering the risk of such disease process have furthered research efforts for identification and development of nutritionally important dietary supplements. Milk is the first complete delivery system for essential nutrients in the newborn. This natural delivery system provides several bioactive components for critica...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A23L1/30A61K38/40C07K14/79
CPCC07K14/79
Inventor NAIDU, A. SATYANARAYAN
Owner NAIDU LP
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